Hi!
I have an issue during tumor dissosciation
During the breast tumor dissociation (human, I use Miltenyi Kit for dossociation) I have poblems with cell counting. Automated cell couter (countess 3 invitrogen) shows me results +- 1.5x10^6 cells/ml, while Burker chamber 100x10^6 cells/ml. What is more pellets of those samples is very, very small, and during staining for mass cytometry I loose most of it. Brucker chamber shows me, that most of my cell population is very small: +-90% (I might be neutrophils? or even smaller).
I have no clue what should I do with it. For staining I need +- 3x10^6 cells in total, but that would means that I will work with unvisible cell pellet.
Does any of you have experience with similar projects?
I assume that you are isolating cells from a mass of dissociated tumor tissue. When you acquire cells in this manner, you have to isolate your target cells and remove the rest in order to count the right type of cell. No use of counting if you have a mixture of cells within your counting chambers. For example, blood cells and epithelial cells might be present in the mixture. If your target cell is epithelial cell, you might want to specifically isolate the epithelial cells only. One way that I have experience of is by using magnetic-activated cell sorting utilizing magnetic beads that are coated with antibodies binding to EPCAM, a biomarker for epithelial cells. To remove blood cells for example leukocytes, you need to use beads that are coated with antibodies targeting CD45 for example. To remove red blood cells, there is a kit to lyse the red blood cells away. In that manner, hopefully, you have concentrated your target cells accurately enough for your counting.
Alternatively, I have seen people using filter membrane/mesh/cell strainer of different pore size if you are aware of the approximate size of your target cells by either collecting the filtrate or the retentate of the filtration. This option of course is less desirable because it is less specific. Please do more reading online for more accurate protocol. The information I provided here is only for a general idea of how things work. Good luck!
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